A failure of immune self-tolerance leads to diseases such as type 1 diabetes, autoimmune thyroiditis, and autoimmune premature ovarian failure. The autoimmune regulator (AIRE) protein is critical to this tolerance. Loss-of-function mutations in AIRE lead to autoimmune polyendocrine syndrome type 1 (APS-1), which is typified by hypoparathyroidism, adrenocortical insufficiency, and mucocutaneous candidiasis and often involves additional endocrine disorders. Aire is expressed by two rare cell populations: medullary thymic epithelial cells (mTECs) and extrathymic Aire-expressing cells (eTACs), which are located in the spleen and lymph nodes. In these cells, Aire promotes expression of tissue-specific proteins, such as insulin. Display of peptides from these proteins permits interaction with autoreactive T cells specific for these antigens, leading to their death, inactivation, or conversion to regulatory T cells. Diminished expression levels of such antigens, including insulin and acetylcholine receptor, have been associated with increased risk of autoimmunity against their tissue of origin. Yet, fifteen years after the discovery of AIRE, the regulation of its expression is poorly understood and the critical cis-regulatory elements (CREs) remain wholly undefined. We have previously accurately recapitulated Aire expression by bacterial artificial chromosome (BAC)-driven GFP-reporter transgenesis. We have also identified a conserved noncoding sequence (CNS) 3 kb upstream of Aire. We propose to investigate how Aire expression is regulated in cis. Specifically, we plan to identify additional candidate Aire CREs by examining public genomic and epigenetic data and by performing ChIP-seq on enhancer-associated histone modifications in Aire-expressing cells (Aim 1) and to use in vivo transgenesis to determine the role of the -3 kb CNS in Aire expression (Aim 2). This research will improve our understanding of the regulation of immune tolerance, will identify potential sites of noncoding APS-1-causing mutations, and will be an early step toward creating mTEC- and eTAC-specific expression cassettes for antigen-specific tolerance induction to treat autoimmune disorders such as type 1diabetes and to prevent transplant rejection.